1. Field of the Invention
This invention relates to an instrument for automatic determination of the base sequence of deoxyribonucleic acid (DNA), particularly to an instrument for determination of the base sequence of DNA which has high resolving power and sensitivity for DNA fragments similar to one another in molecular weight.
2. Related Art Statement
As one example of structure of conventional instrument (a real-time DNA fragment detection type gel electrophoretic instrument) in which DNA fragments in course of gel electrophoresis are detected in real time and the base sequence of DNA is determined thereby, in FIG. 1 is shown the structure of an instrument in which .beta.-ray radiated from DNA fragments in course of gel electrophoresis are detected in real time and the gel electrophoretic pattern is automatically read. In FIG. 1, gel 8 is placed vertically, being held between an electrophoretic plate 6 and an electrophoretic plate 7. Wells 9 for supplying a DNA sample are provided at the upper end of the gel. A buffer solution tank 1 and a buffer solution tank 3 are attached to the upper part and the lower part, respectively, of the gel 8. A buffer solution 2 and a buffer solution 4 are placed in the buffer solution tank 1 and the buffer solution tank 3, respectively, and are in contact with the upper end and the lower end, respectively, of the gel 8. An electric source power 5 is connected to the buffer solution tank 1 and the buffer solution tank 3 and applied a direct current high voltage so that the upper end of the gel 8 becomes a negative electrode.
When a DNA fragment sample labeled with a radioisotope such as .sup.32 P or .sup.35 S is supplied to wells 9 for four kinds of complementary strand synthesis reaction system (A reaction system, C reaction system, G reaction system and T reaction system), respectively, the DNA fragments migrate from the negative electrode towards the positive electrode [F. Sanger et al., "DNA sequencing with chain-terminating inhibitors", Proc. Natl. Acad. Sci. USA, 74, 12, pp. 5463-5467 (1977)]. In this case, the lower the molecular weight of DNA fragment, the higher the migration speed, and DNA fragments having the same molecular weights form a DNA band (a band which DNA fragments having the same molecular weights form on the gel) 10.
In this instrument, slits 13 for detecting .beta.-ray are provided on the side of the electrophoretic plate 6 or the electrophoretic plate 7, and .beta.-ray detectors 14 are attached. When the DNA bands pass in front of the slit 13, .beta.-ray radiated from the DNA band enter the .beta.-ray detector 14. Outputs from the .beta.-ray detectors 14 pass through a signal processing circuit 15 and then enter a computer 16. The computer 16 conducts data processing and determines the base sequence. Although one set of the .beta.-ray detectors 14 are used in this instrument, it is, of course, also possible to use two or more sets.
FIG. 2 is a cross-sectional view showing the structure of the .beta.-ray detector 14 in detail. A scientillator 23 is attached to the end of a photo-multiplier tube 21 through silicone grease 22, and the whole detector is packed in a housing code 24 for shielding from light. The .beta.-ray 25 radiated from the DNA band 10 passes through the slit 13 and is converted into light 26 in the scientillator 23.
The light 26 goes out of the scientillator, passes through the silicone grease 22, and enter the photo-multiplier tube 21, in which it is converted into electric current and detected. A film 27 between the slit 13 and the gel 8 serves for the purpose of insulating the slit 13 from the gel 8 electrically and preventing the slit 13 from being contaminated with the radioisotope in the DNA band 10. The film 27 is made of a material which transmits .beta.-ray (e.g, a polyester film) and has such a thickness as cause no lowering of the detection sensitivity or precision.
In order to elevate the detection sensitivity in a part for detecting .beta.-ray radiated from DNA band, the slit width should be, as shown in FIG. 3, broadened to increase the amount of .beta.-ray which enter the .beta.-ray detector. However, DNA band similar to one another in molecular weight are similar also in distance of migration and overlap in the slit part, resultings in a low resolving power for the DNA bands. On the other band, enhancement of the resolving power for the DNA bands similar in distance of migration by narrowing the slit width leads, as shown in FIG. 4, to a decrease of the amount of .beta.-ray which enters the .beta.-ray detector, and hence to a lowering of the sensitivity. Thus, the conventional instruments are disadvantageous.
In prior art, there have been neither disclosed nor considered control of the temperature of a gel through which DNA fragments migrate by electropheresis, for the purpose of enhancing the resolving power and sensitivity for such DNA bands similar in molecular weight.
As described above, in the conventional instruments for determination of the base sequence of DNA, improvement in the resolving power and sensitivity for DNA fragments similar in molecular weight has been neither disclosed nor taken into consideration, and the sensitivity is low in determination of the base sequences of DNA fragments similar in molecular weight. Thus, the conventional instruments have been not sufficiently satisfactory.